The nasal cavity is an important niche for Staphylococcus aureus. Young children-particularly those age 3 or younger-are more likely to be colonized by S. aureus than older children or adults. S. aureus nasal colonization significantly increases the risk for subsequent infection, but decolonization strategies for children are limited, and mupirocin resistance is emerging. In recent years, S. aureus infection rates have also steadily risen in the pediatric population, despite opposing trends in adults. Better preventative strategies against S. aureus are urgently needed to reduce pediatric infections. Our long-term goal is to develop probiotic-based strategies to decrease S. aureus colonization, transmission, and infection in children. To achieve this, we need to better understand the dynamics and determinants of pediatric nasal microbiota. The overall objective of this proposal is to identify commensals that can competitively exclude S. aureus in the pediatric nasal environment. The central hypothesis, based on our preliminary studies, is that competitive exclusion from non-pathogenic nasal commensals, such as Dolosigranulum, can persistently resist S. aureus colonization. The rationale for the proposed research is that a better understanding of nasal microbiota stability and succession, combined with experimental testing of how Dolosigranulum and other commensals interact with S. aureus will provide important insights into the potential effectiveness of nasal probiotics. The study's hypothesis wil be tested through two specific aims: Aim 1: Determine if children with high absolute abundances of specific nasal commensals have lower risk of carrying S. aureus in the first year of life; and Aim 2: Empirically determine Dolosigranulum's ability to competitively exclude S. aureus in a novel epithelium-microbiome culture model. Aim 1 will be accomplished using an existing collection of longitudinal nasal samples from infants in the first year of life (n = 203). The baseline (i.e., month 1) sample will be characterized by absolute abundance-based microbiome analysis to identify infants with Dolosigranulum- or S. aureus-dominated nasal community state type (CST). The microbial succession of 30 infants with each CST at baseline will be characterized to determine the dynamics of these CSTs. To assess nasal bacterial competition experimentally, Aim 2 will construct an epithelium-microbiome model using samples with select CSTs from Aim 1, which will undergo direct challenges with S. aureus and other nasal CSTs. The approach is innovative, in this team's opinion, because it will construct and validate a novel in vitro model system to recapitulate observed nasal CSTs and enable empiric testing of our competitive-exclusion hypothesis in a mixed culture environment. The proposed research is significant, because it is a proof-of-principle study of nasal probiotics against S. aureus. Importantly, the project will generate an effective model system for future host-microbe and microbe-microbe studies that will accelerate the discovery of new ways to prevent S. aureus colonization and infection.